Pharmacological activities of Vitex agnus-castus extracts in vitro.

The pharmacological effects of ethanolic Vitex agnus-castus fruit-extracts (especially Ze 440) and various extract fractions of different polarities were evaluated both by radioligand binding studies and by superfusion experiments. A relative potent binding inhibition was observed for dopamine D2 and opioid (micro and kappa subtype) receptors with IC50 values of the native extract between 20 and 70 mg/mL. Binding, neither to the histamine H1, benzodiazepine and OFQ receptor, nor to the binding-site of the serotonin (5-HT) transporter, was significantly inhibited. The lipophilic fractions contained the diterpenes rotun-difuran and 6beta,7beta-diacetoxy-13-hydroxy-labda-8,14-dien . They exhibited inhibitory actions on dopamine D2 receptor binding. While binding inhibition to mu and kappa opioid receptors was most pronounced in lipophilic fractions, binding to delta opioid receptors was inhibited mainly by a aqueous fraction. Standardised Ze 440 extracts of different batches were of constant pharmacological quality according to their potential to inhibit the binding to D2 receptors. In superfusion experiments, the aqueous fraction of a methanolic extract inhibited the release of acetylcholine in a concentration-dependent manner. In addition, the potent D2 receptor antagonist spiperone antagonised the effect of the extract suggesting a dopaminergic action mediated by D2 receptor activation. Our results indicate a dopaminergic effect of Vitex agnus-castus extracts and suggest additional pharmacological actions via opioid receptors.     

Hippocampal astrocytes exhibit Ca2+-elevating muscarinic cholinergic and histaminergic receptors in situ

Recent findings suggest that astrocytes respond to neuronally released neurotransmitters with Ca2+ elevations. These Ca2+ elevations may trigger astrocytes to release glutamate, affecting neuronal activity. Neuronal activity is also affected by modulatory neurotransmitters that stimulate G protein-coupled receptors. These neurotransmitters, including acetylcholine and histamine, might affect neuronal activity by triggering Ca2+-dependent release of neurotransmitters from astrocytes. However, there is no physiological evidence for histaminergic or cholinergic receptors on astrocytes in situ. We asked whether astrocytes have these receptors by imaging Ca2+-sensitive dyes sequestered by astrocytes in hippocampal slices. Our results show that immunocytochemically identified astrocytes respond to carbachol and histamine with increases in intracellular free Ca2+ concentration. The H1 histamine receptor antagonist chlorpheniramine inhibited responses to histamine. Similarly, atropine and the M1-selective muscarinic receptor antagonist pirenzepine inhibited carbachol-elicited responses. Astrocyte responses to histamine and carbachol were compared with responses elicited by alpha1-adrenergic and metabotropic glutamate receptor agonists. Individual astrocytes responded to different subsets of receptor agonists. Ca2+ oscillations were the prevalent response pattern only with metabotropic glutamate receptor stimulation. Finally, functional alpha1-adrenergic receptors and muscarinic receptors were not detected before postnatal day 8. Our data show that astrocytes have acetylcholine and histamine receptors coupled to Ca2+. Given that Ca2+ elevations in astrocytes trigger neurotransmitter release, it is possible that these astrocyte receptors modulate neuronal activity.     

Adhyperforin as a contributor to the effect of Hypericum perforatum L. in biochemical models of antidepressant activity

The present paper describe investigations which demonstrate that hyperforin is not the only phloroglucinol derivative in extracts of the medicinal plant Hypericum perforatum L., which possess a biological activity. Hyperforin was the major lipophilic constituent in two different extracts, whereas the amount of adhyperforin was approximately 10 times lower. Adhyperforin, like hyperforin, is a potent inhibitor of the uptake of dopamine, serotonin and noradrenaline. Neither hyperforin nor adhyperforin inhibited binding of the cocaine analogue, [3H]WIN 35,428 to the dopamine transporter. However, the known antidepressives imipramine, nomifensine and fluoxetine all inhibited binding of [3H]WIN 35,428, indicating that hyperforin and adhyperforin do not bind to the same site on the dopamine transporter as these compounds. Furthermore, hyperforin and adhyperforin did not prevent dopamine binding, but inhibited dopamine translocation. Our studies further support recent reports suggesting that the effect of hyperforin on uptake of monoamines is probably not caused by a direct effect of hyperforin on known sites on the transporters.     

GABAA Receptor Complex in an Experimental Model of Hepatic Encephalopathy: Evidence for Elevated Levels of an Endogenous Benzodiazepine Receptor Ligand

The involvement of the gamma-aminobutyric acidA (GABAA) receptor complex in the pathogenesis of hepatic encephalopathy was examined in thioacetamide-treated rats with fulminant hepatic failure. Partially purified extracts from encephalopathic rat brain were approximately three times more potent in inhibiting [3H]Ro 15-1788 binding to benzodiazepine receptors than identically prepared extracts from control rats. High levels of inhibitory activity were also found in extracts of plasma, heart, and liver from thioacetamide-treated rats. The inhibition of [3H]Ro 15-1788 binding by brain extracts appeared to be competitive and reversible and was unaffected by treatment with either proteolytic enzymes or boiling. Further, GABA significantly enhanced the potency of these extracts in inhibiting [3H]flunitrazepam binding. In contrast, no differences were found in radioligand binding to the constituent recognition sites of the GABAA receptor complex in well-washed brain membranes prepared from control and encephalopathic animals. These findings suggest that the recognition-site qualities of the constituent proteins of the GABAA receptor complex are unchanged in an experimental model of hepatic encephalopathy. However, significant elevations in the level of a substance or substances with neurochemical properties characteristic of a benzodiazepine receptor agonist may contribute to the electrophysiological and behavioral manifestations of hepatic encephalopathy.     

Novel ligands for the human histamine H1 receptor: synthesis, pharmacology, and comparative molecular field analysis studies of 2-dimethylamino-5-(6)-phenyl-1,2,3,4-tetrahydronaphthalenes

This paper reports the synthesis of a novel series of (+/-)-2-dimethylamino- 5- and 6-phenyl-1,2,3,4-tetrahydronaphthalene derivatives (5- and 6-APTs), and, corresponding affinity, functional activity, and, molecular modeling studies with regard to drug design targeting the human histamine H1 receptor. The 5-APTs have 2- to 4-fold higher H1 receptor affinity than the endogenous agonist histamine. The chemical nature of a meta-substituent on the 5-APT pendant phenyl moiety does not significantly affect H1 affinity. In contrast, analogous meta-substitution for the 6-APTs increases H1 affinity up to 100-fold. The new APTs do not activate H1 receptor-linked intracellular signaling and apparently are competitive H1 antagonists. A new model that establishes structural parameters for binding to the human H1 receptor by APTs and other ligands was developed using 3-D QSAR (CoMFA). The model predicts H1 ligand binding with a higher degree of external predictability compared to a previously reported model. The APTs also were examined for activity at human serotonin 5-HT2A and 5-HT2C receptors, which are phylogenetically closely related to the H1 receptor. 5-APT and m-Cl-6-APT were identified as novel agonists that selectively activate 5-HT2C receptors. It is concluded that the lipophilic (brain-penetrating) APT molecular scaffold may have pharmacotherapeutic potential in neuropsychiatric diseases.     

Evidence of fast serotonin transmission in frog slowly adapting type 1 responses

The Merkel cell-neurite (MCN) complex generates slowly adapting type 1 (SA1) response when mechanically stimulated. Both serotonin (5-HT) and glutamate have been implicated in the generation of normal SA1 responses, but previous studies have been inconclusive as to what their roles are or how synaptic transmission occurs. In this study, excised dorsal skin patches from common water frogs (Rana ridibunda) were stimulated by von Frey hairs during perfusion in a tissue bath, and single-unit spike activity was recorded from SA1 fibres. Serotonin had no significant effect on the SA1 response at low (10?μM) concentration, significantly increased activity in a force-independent manner at 100?μM, but decreased activity with reduced responsiveness to force at 1?mM. Glutamate showed no effect on the responsiveness to force at 100?μM. MDL 72222 (100?μM), an ionotropic 5-HT3 receptor antagonist, completely abolished the responsiveness to force, suggesting that serotonin is released from Merkel cells as a result of mechanical stimulation, and activated 5-HT3 receptors on the neurite. The metabotropic 5-HT2 receptor antagonist, ketanserin, greatly reduced the SA1 fibre's responsiveness to force, as did the non-specific glutamate receptor antagonist, kynurenic acid. This supports a role for serotonin and glutamate as neuromodulators in the MCN complex, possibly by activation and/or inhibition of signalling cascades in the Merkel cell associated with vesicle release. Additionally, it was observed that SA1 responses contained a force-independent component, similar to a dynamic response observed during mechanical vibrations.     

Glutamate Inhibits Adenylate Cyclase Activity in Dispersed Rat Hippocampal Cells Directly via an N-Methyl-d-Aspartate-Like Metabotropic Receptor

Three major subtypes of glutamate receptors that are coupled to cation channels--N-methyl-D-aspartate (NMDA), kainate, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors--are known as ionotropic receptors in the mammalian CNS. Recently, an additional subtype that is coupled to GTP binding proteins and stimulates (or inhibits) metabolism of phosphoinositides has been proposed as a metabotropic receptor. Incubation of dispersed hippocampal cells from adult rats with glutamate or NMDA decreased forskolin-stimulated cyclic AMP (cAMP) accumulation; half-maximal effects were obtained with 5.6 +/- 2.2 and 6.4 +/- 2.3 microM, respectively. Kainate and quisqualate were less potent. The effect of glutamate was antagonized by 2,3-diaminopropionate and 2-amino-5-phosphonovalerate, NMDA/glutamate receptor antagonists, but not by 0.5 microM Joro spider toxin, a specific blocker of the AMPA receptor. The inhibitory effect of glutamate on cAMP formation was not blocked by 2 microM tetrodotoxin or by the absence of Ca2+. In hippocampal membranes, glutamate, similar to carbachol, inhibited adenylate cyclase activity in a GTP-dependent manner. These findings suggest that the glutamate inhibition of adenylate cyclase is direct and is not due to a result of the release of other neurotransmitters. The effect of glutamate on cAMP accumulation was observed in an assay medium containing 0.7 mM MgCl2, which is known to inhibit both ionotropic NMDA receptor/channels in the hippocampus and metabotropic NMDA receptors in the cerebellum. The inhibitory effect of glutamate was abolished by pertussis toxin treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

鲫鱼脑氨基酸类神经递质受体在两栖类卵母细胞中的表达

两栖类卵母细胞表达系统经注射鲫鱼脑ｍＲＮＡ后可表达多种神经递质受体和某些离子通道。本工作利用电压箝方法结合药理学手段对ＧＡＢＡ受体和谷氨酸离子型受体作了较详细的研究。结果表明,由ＧＡＢＡ诱发的电流反应中,约９０％由ＧＡＢＡＡ受体介导,乘余约１０％的成分对ＧＡＢＡＡ受体的专一性拮抗剂Ｂｉｃｕｃｕｌｌｉｎｅ不敏感,而ＧＡＢＡＢ受体的专一性激动剂Ｂａｃｌｏｆｅｎ不能引进电流反应,因此这部分受体特性与ＧＡ     

Serotonin 5-HT(7) receptors mediate relaxation of porcine pial veins

Isolated porcine pial veins in the presence of active muscle tone have been shown to exhibit rhythmic contractions (RC) that are inhibited by serotonin (5-HT) in a concentration-dependent manner. The 5-HT inhibition of RC is mediated by an as yet unidentified 5-HT receptor subtype located on the vascular smooth muscle. 5-carboxamidotryptamine, which is a potent but nonselective agonist at 5-HT(7) receptors, has been shown to be the most potent inhibitor of RC in porcine pial veins. Therefore, the present study was designed to determine if the 5-HT-mediated inhibition of RC in pial veins is mediated by 5-HT(7) receptors and if 5-HT(7) receptor mRNA is expressed in endothelium-denuded pial veins; the study was done with the use of an in vitro tissue bath and RT-PCR techniques. Our findings indicated that 5-HT inhibition of RC in porcine pial veins was prevented by 5-HT(7)-receptor antagonists (clozapine, pimozide, and LY-215840) in a concentration-dependent manner. Furthermore, a strong PCR signal for the 5-HT(7) receptor was consistently detected in endothelium-denuded pial veins. Sequence analysis of the amplified products confirmed their high degree of homology with the porcine and/or human 5-HT(7)-receptor gene. Taken together, these data suggest that the 5-HT-induced inhibition of RC in porcine pial veins is at least in part mediated by 5-HT(7) receptors located on the venous smooth muscle.     

Binding of antipsychotic drugs to human brain receptors focus on newer generation compounds

Using radioligand binding assays and post-mortem normal human brain tissue, we obtained equilibrium dissociation constants (K(d)s) for nine new antipsychotic drugs (iloperidone, melperone, olanzapine, ORG 5222, quetiapine, risperidone, sertindole, ziprasidone, and zotepine), one metabolite of a new drug (9-OH-risperidone), and three older antipsychotics (clozapine, haloperidol, and pimozide) at nine different receptors (alpha1-adrenergic, alpha2-adrenergic, dopamine D2, histamine H1, muscarinic, and serotonin 5-HT1A, 5-HT1D, 5-HT2A, and 5-HT2C receptors). Iloperidone was the most potent drug at the two adrenergic receptors. ORG 5222 was the most potent drug at dopamine D2 and 5-HT2c receptors, while ziprasidone was the most potent compound at three serotonergic receptors (5-HT1A, 5-HT1D, and 5-HT2A). At the remaining two receptors, olanzapine was the most potent drug at the histamine H1 receptor (Kd=0.087 nM); clozapine at the muscarinic receptor (Kd=9 nM). Certain therapeutic and adverse effects, as well as certain drug interactions can be predicted from a drug's potency for blocking a specific receptor. These data can provide guidelines for the clinician in the choice of antipsychotic drug.     

Role of serotonin,gamma-aminobutyric acid,and glutamate in the behavioral thermoregulation of female tilapia during the prespawning phase

The role of hypothalamic neurotransmitter systems in behavioral thermoregulation was investigated in the prespawning female tilapia, Oreochromis mossambicus. Intrahypothalamic microinjection with serotonin (5-HT, 3 microliters of 1.0 x 10(-6) M) resulted in a significant increase in the selected temperature. This effect was mimicked by the agonist of 5-HT1A, 1B, and 2C receptors, N-3-trifluoromethylphenyl piperazine. Intrahypothalamic microinjection of tilapia with gamma-aminobutyric acid (GABA) resulted in a biphasic effect of the temperature selection, whereas microinjection with muscimol, an agonist of GABAA receptor, had no effect on temperature selection. Both agonist and antagonist of glutamate (Glu), N-methyl-D-aspartate (NMDA), and MK-801 (1.0 x 10(-6) M), a noncompetitive blocker of NMDA receptor, significantly decreased the preferred temperature. These results indicate that the hypothalamic 5-HT, GABA, and Glu systems play a role in the temperature selection of prespawning female tilapia.     

Receptor-independent depression of DA and 5-HT uptake by cannabinoids in rat neocortex--involvement of Na(+)/K(+)-ATPase

There is evidence that cannabinoids modulate the reuptake of some neurotransmitters in the central nervous system. In this study, we investigated the effects of the synthetic cannabinoid receptor agonist WIN55212-2, the endocannabinoid anandamide and the chemically related arachidonic acid on serotonin (5-HT) and dopamine (DA) uptake into rat neocortical synaptosomes. At micromolar concentrations, anandamide and arachidonic acid produced steep inhibition curves with Hill coefficients above unity. WIN55212-2 inhibited both DA and 5-HT uptake with Hill coefficients near unity, also within the micromolar range. The effect of WIN55212-2 was not mediated by cannabinoid receptors, since the CB1 receptor antagonist AM251 failed to diminish uptake inhibition by WIN55212-2 and since the Ki estimates of WIN55212-2 were outside the range of the dissociation constants of WIN55212-2 at both CB1 and CB2 receptors. A 100-fold higher concentration of DA, respectively 5-HT, did not induce a shift to the right of the WIN55212-2 concentration-inhibition curves, suggesting a carrier-independent mechanism. The Na(+)/K(+)-ATPase inhibitor ouabain concentration dependently inhibited 5-HT uptake. Possible drug effects on commercial Na(+)/K(+)-ATPase and synaptosomal ATP consumption were investigated using an ATP bioluminescence assay. Ouabain inhibited both commercial and synaptosomal Na(+)/K(+)-ATPase. WIN55212-2 had no effect on commercial Na(+)/K(+)-ATPase, but inhibited synaptosomal ATP consumption. Anandamide produced a sharp decrease in the activity of commercial Na(+)/K(+)-ATPase and on synaptosomal ATP consumption. Presence of ouabain significantly reduced the inhibitory effect of anandamide on synaptosomal ATP consumption, whereas the effect of WIN55212-2 remained unchanged. Our results show that cannabinoids and arachidonic acid inhibit DA and 5-HT uptake into rat neocortical synaptosomes. This effect is neither cannabinoid receptor-mediated nor due to competitive inhibition of membrane transporters, but is partly effected by a decreased Na(+)/K(+)-ATPase activity.     

N1-Methyl-2-125I-Lysergic Acid Diethylamide, a Preferred Ligand for In Vitro and In Vivo Characterization of Serotonin Receptors

Methylation of 2-125I-lysergic acid diethylamide (125I-LSD) at the N1 position produces a new derivative, N1-methyl-2-125I-lysergic acid diethylamide (125I-MIL), with improved selectivity and higher affinity for serotonin 5-HT2 receptors. In rat frontal cortex homogenates, specific binding of 125I-MIL represents 80-90% of total binding, and the apparent dissociation constant (KD) for serotonin 5-HT2 receptors is 0.14 nM (using 2 mg of tissue/ml). 125I-MIL also displays a high affinity for serotonin 5-HT1C receptors, with an apparent dissociation constant of 0.41 nM at this site. 125I-MIL exhibits at least 60-fold higher affinity for serotonin 5-HT2 receptors than for other classes of neurotransmitter receptors, with the dopamine D2 receptor as its most potent secondary binding site. Studies of the association and dissociation kinetics of 125I-MIL reveal a strong temperature dependence, with very slow association and dissociation rates at 0 degree C. Autoradiographic experiments confirm the improved specificity of 125I-MIL. Selective labeling of serotonin receptors was observed in all brain areas examined. In vivo binding studies in mice indicate that 125I-MIL is the best serotonin receptor label yet described, with the highest frontal cortex to cerebellum ratio of any serotonergic radioligand. 125I-MIL is a promising ligand for both in vitro and in vivo labeling of serotonin receptors in the mammalian brain.     

Effects of sesquiterpenoids from "Oriental incenses" on acetic acid-induced writhing and D2 and 5-HT2A receptors in rat brain.

Six sesquiterpenoids, namely jinkoh-eremol, agarospirol, alpha- and beta-santalols, dehydrocostus lactone and costunolide, isolated from oriental incenses inhibited acetic acid-induced writhing in mice. The incidence of writhing produced by jinkoh-eremol, alpha-santalol and costunolide were revealed by administration of naloxone (mu-, kappa- and delta-antagonists). Inhibitory activities of alpha-santalol on opioid receptors were shown only by the delta antagonist, but not by the mu- and kappa-antagonists. The delta2-antagonist, but not the delta-antagonist, inhibited the activity of alpha santalol. The mechanism of inhibitory activity on the opioid receptor by alpha-santalol was different from that of morphine. Alpha-santalol was shown to be the most potent of the six as an antagonist of dopamine D2 and serotonine 5-HT2A receptor binding. The effect of alpha-santalol, was the same as that of chlorpromazine as an antipsychotic agent, although alpha-santalol was less potent than chlorpromazine.     

Evaluation of FLIPR Calcium 3 Assay Kit--a new no-wash fluorescence calcium indicator reagent

We have evaluated the FLIPR Calcium 3 Assay Kit (Calcium 3), a new no-wash fluorescence calcium indicator dye reagent, for the measurement of agonist-stimulated calcium signaling in cells expressing the serotonin 2C (5-HT(2C)), metabotropic glutamate receptor 5 (mGluR5) and the vasopressin 2 (V2) G-protein-coupled receptors. Calcium 3 yielded equivalent (5-HT(2C)) or superior (mGluR5 and V2) sensitivity to FLUO-4 as indexed by the change in fluorescence counts following agonist application. Assay variability, indexed by CV, using Calcium 3 or FLUO-4 was equivalent with 5-HT(2C) receptor responses although CVs were reduced using Calcium 3 in the examples of the mGluR5 and V2 receptors. Receptor pharmacologies based on agonist EC(50) values were identical when either Calcium 3 or FLUO-4 were utilized. Our results validate Calcium 3 as a compelling alternative to FLUO-4 in the choice of fluorescent dye reagent for studying G-protein-coupled receptors, providing the advantage of a homogenous, no-wash assay format.     

Serotomide and safflomide modulate forskolin-stimulated cAMP formation via 5-HT1 receptor

Serotomide (trans-N-caffeoylserotonin) and safflomide (trans-N-caffeoyltryptamine) belong to serotonin-derived phenylpropenoid amides found in plants. In this paper, serotomide and safflomide were investigated to determine their effects on serotonin receptor 5-HT1 in the renal epithelial (OK) cells, due to their structural similarity to 5-HT1 receptor ligands. At the concentration of 10 μM, serotomide was able to inhibit forskolin-stimulated cAMP formation in the OK cells by 31% (p<0.019). The inhibition was repressed by Nan-190 and spiperone (5-HT1 antagonists), suggesting that serotomide suppresses cAMP formation via binding to 5-HT1 receptors in the OK cells. Meanwhile, safflomide could not inhibit forskolin-stimulated cAMP formation at the same concentration (10 μM), but repress the inhibition of forskolin-stimulated cAMP by serotonin agonists (e.g., serotonin and 8-OH-DPAT) by 31% (p<0.018), suggesting that safflomide may block 5-HT1 receptors in a similar way to Nan-190 and spiperone. All together the data indicate that serotomide and safflomide may be potent compounds that respectively act to activate and to block 5-HT1 receptors on OK cells.     

The antidepressant mechanism of Hypericum perforatum

Clinical data indicate that hydroalcoholic extracts of Hypericum perforatum might be as valuable as conventional antidepressants in mild-to-moderate depression, with fewer side effects. One clinical trial using two extracts with different hyperforin contents indicated it as the main active principle responsible for the antidepressant activity. Behavioural models in rodents confirm the antidepressant-like effect of Hypericum extracts and also of pure hyperforin and hypericin. A hydroalcoholic extract lacking hyperforin also lacks the antidepressant-like effect. According to pharmacokinetic data and binding studies, it appears that the antidepressant effect of Hypericum extract is unlikely be due to an interaction of hypericin with central neurotransmitter receptors. The main in vitro effects of hyperforin (at concentrations of 0.1-1 microM) are non-specific presynaptic effects, resulting in the non-selective inhibition of the uptake of many neurotransmitters, and the interaction with dopamine D1 and opioid receptors. However, it is still not clear whether these mechanisms can be activated in vivo, since after administration of Hypericum extract brain concentrations of hyperforin are well below those active in vitro. In the rat, Hypericum extract might indirectly activate sigma receptors in vivo (through the formation of an unknown metabolite or production of an endogenous ligand), suggesting a new target for its antidepressant effects.     

A serotonin receptor antagonist induces oocyte maturation in both frogs and mice: evidence that the same G protein-coupled receptor is responsible for maintaining meiosis arrest in both species

Accumulating evidence has indicated that vertebrate oocytes are arrested at late prophase (G2 arrest) by a G protein coupled receptor (GpCR) that activates adenylyl cyclases. However, the identity of this GpCR or its regulation in G2 oocytes is unknown. We demonstrated that ritanserin (RIT), a potent antagonist of serotonin receptors 5-HT2R and 5-HT7R, released G2 arrest in denuded frog oocytes, as well as in follicle-enclosed mouse oocytes. In contrast to RIT, several other serotonin receptor antagonists (mesulergine, methiothepine, and risperidone) had no effect on oocyte maturation. The unique ability of RIT, among serotonergic antagonists, to induce GVBD did not match the antagonist profile of any known serotonin receptors including Xenopus 5-HT7R, the only known G(s)-coupled serotonin receptor cloned so far in this species. Unexpectedly, injection of x5-HT7R mRNA in frog oocytes resulted in hormone-independent frog oocyte maturation. The addition of exogenous serotonin abolished x5-HT7R-induced oocyte maturation. Furthermore, the combination of x5-HT7R and exogenous serotonin potently inhibited progesterone-induced oocyte maturation. These results provide the first evidence that a G-protein coupled receptor related to 5-HT7R may play a pivotal role in maintaining G2 arrest in vertebrate oocytes.     

Serotonin 5-HT1D and 5-HT1A Receptors Respectively Mediate Inhibition of Glutamate Release and Inhibition of Cyclic GMP Production in Rat Cerebellum In Vitro

Abstract: The K⁺-evoked overflow of endogenous glutamate from cerebellar synaptosomes was inhibited by serotonin [5-hydroxytryptamine (5-HT); pD₂ = 8.95], 8-hydroxy-2-(di- n -propylamino)tetralin (8-OH-DPAT; pD₂ = 7.35), and sumatriptan (pD₂ = 8.43). These inhibitions were prevented by the selective 5-HT_1D receptor antagonist N -[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)(1,1-biphenyl)-4-carboxamide (GR-127935). The three agonists tested also inhibited the cyclic GMP (cGMP) response provoked in slices by K⁺ depolarization; pD₂ values were 9.37 (5-HT), 9.00 (8-OH-DPAT), and 8.39 (sumatriptan). When cGMP formation was elevated by directly activating glutamate receptors with NMDA or α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), the inhibition of the cGMP responses displayed the following pattern: 5-HT (pD₂ values of 8.68 and 8.72 against NMDA and AMPA, respectively); 8-OH-DPAT (respective pD₂ values of 9.15 and 9.00); sumatriptan (0.1 µ M ) was ineffective. The 5-HT_1A receptor antagonist ( S )-(+) N-tert -butyl-3-[4-(2-methoxyphenyl)piperazin-1-yl]-2-phenylpropionamide dihydrochloride [(+)-WAY 100135] did not prevent the inhibition of glutamate release by 5-HT but blocked the inhibition by 8-OH-DPAT of the NMDA/AMPA-evoked cGMP responses. It is suggested that presynaptic 5-HT_1D receptors mediate inhibition directly of glutamate release and indirectly of the cGMP responses to the released glutamate; on the other hand, activation of (postsynaptic) 5-HT_1A receptors causes inhibition of the cGMP responses linked to stimulation of NMDA/AMPA receptors.     

Towards a view of functioning dimeric metabotropic receptors

X-ray crystallography was used to solve the atomic structure of the ligand binding domain of the metabotropic glutamate receptor type1 homo-dimer, making it possible to show the conformational change of this domain upon glutamate binding. Studies of dimeric metabotropic receptors thereafter have focused on the respective roles and interaction of the two subunits, on the activation mechanisms following the structural rearrangements of the ligand-binding domain, and on the functional significance of polyvalent cations, the binding of which was identified in the crystal. The direct interaction between the GABA(B) receptor and the metabotropic glutamate receptor (mGluR1) has also attracted attention. Recently, attention has focused on incorporating these structural features into a functional view of the receptors.